1. Field of Invention
This invention relates to a non-naturally occurring lipoprotein composite containing an MRI contrast agent or a therapeutic substance in its core.
2. Description of Related Art
Lipoproteins are macromolecular composites formed by lipids and proteins at different ratios, sizes and densities. Lipoproteins transport water-insoluble lipids (e.g., cholesterol) in the blood. Lipoproteins comprise an apolar core surrounded by a phospholipid monolayer containing unesterified cholesterol and apolipoproteins. The five main lipoprotein classes include chylornicrons (75-1200 nm), very-low-density lipoprotein (30-80 nm), intermediate-low-density lipoprotein (25-35 nm), low-density lipoprotein (LDL) (18-25 nm) and high-density lipoprotein (HDL) (8-12 nm) (see Vance, D. E., Vance J E (eds.) (1996) Elsevier 31).
Lipoproteins are good candidates for drug delivery or imaging because they are not recognized as foreign entities by the human immune system and escape absorption by the reticuloendothelial system (see U.S. Pat. No. 5,948,756 to Barenholz et al.).
Zheng, et al. studied rerouting lipoprotein nanoparticles to selected alternate receptors for the targeted delivery of cancer diagnostic and therapeutic agents (see PNAS 102[40] 17752-17762 (2005)). A lipoprotein-based nanoplatform (LBNP) was generated by conjugating tumor homing molecules to the protein components of naturally occurring lipoproteins, wherein a low-density lipoprotein (LDL) folate receptor (FR)-targeted agent was prepared by conjugating folic acid to lysine residues of the apoB-100 protein, which is an apolipoprotein. The article describes reconstituting the lipid core reconstituted with a lipophilic photodynamic therapy agent tetra-t-butyl-silicon phthalocyanine bisoleate (SiPc-BOA).
An example of natural lipoprotein is low-density lipoprotein (LDL). The low-density lipoprotein (LDL) particle contains a lipid core of some 1500 esterified cholesterol molecules and triglycerides. A shell of phospholipids and unesterified cholesterol surrounds this highly hydrophobic core. The shell also contains a single copy of apoB-100, which is recognized by the LDL receptor (LDLR).
Zheng, et al. developed various reconstituted LDL, i.e., natural LDL with a modified core/shell to incorporate imaging agents. Another method developed by Zheng, et al. is to form a phospholipid micelle that is further decorated by an apolipoprotein (or apoprotein, used interchangeably hereafter), which renders it LDL-like. These methods can be extended beyond LDL to other members of the cholesterol family. The advantage of LDL or LDL-like entity as a delivery vehicle lies in its intrinsic biocompatibility and richness of variety. The surface of the entity can be further modified, by ligand or antibody, to make it target-specific.
International Application Publication No. WO 2006/073419 to Zheng et al. discloses non-naturally occurring lipoprotein nanoplatforms (“LBNP”) that allow targeted delivery of active agents and can be used to create a diverse set of multifunctional cancer diagnostic and therapeutic devices.
Diverse targeting is achieved by conjugating certain tumor-homing molecules (e.g., folic acid) to the Lys residues exposed on the apoB-100 surface optionally followed by capping the remaining unreacted Lys residues. LDLR binding is turned off and the modified LDL particles are redirected to the desired cancer signatures and/or specific tissues, i.e., molecules that are selectively overexpressed in various types of cancer cells. In particular embodiments, the multifunctionality of LBNP provides targeted delivery of active agents e.g., diagnostic and/or therapeutic agents. Such diagnostic agents include magnetic resonance imaging (MRI) agents, near-infrared fluorescence (NIRF) probes and photodynamic therapy (PDT) agents.
High-resolution contrast enhanced magnetic resonance imaging (MRI) is one of the most useful techniques for screening tumors and other anatomical abnormalities. In MRI, an image of an organ or tissue is obtained by placing a subject in a strong magnetic field and observing the interactions between the magnetic spins of the protons and radiofrequency electromagnetic radiation.
Due to sensitivity limitation of the current MRI techniques, efficient recognition requires a very high capacity target like fibrin, which is present in sufficient quantity to be seen with simple targeted Gd chelates, or targets accessible to the blood stream that can be bound with a Gd cluster, polymer or an iron particle. This is possible presently only in a limited target set. For example, the seminal work by Sipkins et al. (Sipkins, D. A. et al. ICAM-i expression in autoimmune encephalitis visualized using magnetic resonance imaging. J Neuroimmunol 104, 1-9 (2000)) demonstrated that paramagnetic immunoliposomes targeted to the integrin receptor, intercellular adhesion molecule-i (ICAM-1), could be used to visualize altered ICAM-1 expression in autoimmune encephalitis using MRI. Lanza and Wickline et al. (Anderson, S. A. et al., Magnetic Resonance in Medicine. 2000 September; 44(3):433-9) developed a fibrin-targeted paramagnetic nanoparticle contrast agent for high-resolution MRI characterization of human thrombus. In their approach, the contrast agent is a lipid-encapsulated perfluorocarbon nanoparticle with numerous Gd-DTPA complexes incorporated into the outer surface. The nanoparticles themselves provide little or no blood-pool contrast when administrated in vivo, but when they bind and collect at a targeted site, such as a thrombus, they modify the TI-contrast of the tissue substantially. Thus, they inherently yield high signal-to-noise ratios. However, unlike imaging fibrin, an extra-cellular target, intracellular MRI imaging is particularly challenging because the minimum concentration of MRI agents required for the MRI detection limit is much higher (about 1 mM) than the extracellular target (40 μM).
WO 2006/073419 describes that diagnostic agents can be associated with a surface or a core of lipoprotein particles LBNP. Diagnostic agents such as MRI contrast agents associated with the inside of the core of LBNP are obtained by replacing cholesterol esters located inside the lipid core with lipophilic agents. WO 2006/073419 does not describe forming a core comprising a lipophobic or a hydrophilic diagnostic agent encapsulated with an amphiphilic cholesterol.
International Application Publication No. WO90/01295 to Menz et al. discloses magnetic resonance (MR) contrast agents associated with ligands which are recognized by receptor mediated endocytosis (RME). MR contrast agents are prepared by co-precipitation of superparamagnetic metal oxides with the ligands, direct conjugation with a ligand or conjugation of a ligand to a silanized superparamagnetic material.
Methods of making of lipophilic agent are described by U.S. Pat. No. 6,645,463B1 to Counsell, U.S. Pat. No. 4,647,445 to Lees, and U.S. Pat. No. 4,452,773 Molday.
Other related technologies and background are described in the following publications: M. Hammel, P. Laggner and R. Prassl “Structural characterization of nucleoside loaded low density lipoprotein as a main criterion for the applicability as drug delivery system”, Chem. Phys. Lipid. 123, 103-207 (2003); R. C. Pittman, et al. “Synthetic High Density Lipoprotein Particles”, J. Bio. Chem. 262[6] 2435-2442 (1987); S. Sun and H. Zeng “Size-controlled synthesis of magnetite nanoparticles”, J. Am. Chem. Soc., 124, 8204-8205; M. Krieger, et al. “Reconstituted low density Lipoprotein: a vehicle for the delivery of hydrophobic fluorescent probes to cells”, JSS 10, 467-478 (1979).
However, despite the current developments, there is still a need in the art to produce an engineered lipoprotein having a core with substance incorporated therein which are not limited to lipophilic substances.
All references cited herein are incorporated herein by reference in their entireties.